Developmental and reproductive toxicity assessment in rats with KGC-HJ3, Korean Red Ginseng with Angelica gigas and Deer antlers.

BACKGROUND: Korean Red Ginseng has been widely used in traditional oriental medicine for a prolonged period, and its pharmacological effects have been extensively investigated. In addition, Angelica gigas and deer antlers were also used as a tonic medicine with Korean Red Ginseng as the oriental herbal therapy. METHODS: This study was conducted to evaluate the potential toxicological effect of KGC-HJ3, Korean Red Ginseng with angelica gigas and deer antlers, on reproductive and developmental functions including fertility, early embryonic development, maternal function, and embryo-fetal development. KGC-HJ3 was administered by oral gavage to Sprague-Dawley rats (22 animals per sex per group) at dose levels of 0 mg/kg (control), 500 mg/kg, 1000 mg/kg, and 2000 mg/kg to evaluate the potential toxicological effect on fertility and early embryonic development. In addition, KGC-HJ3 was also administered by oral gavage to mating-proven Sprague-Dawley rats (22 females per group) during the major organogenesis period at dose levels of 0 mg/kg (control), 500 mg/kg, 1000 mg/kg, and 2000 mg/kg to evaluate the potential toxicological effect on maternal function and embryo-fetal development. RESULTS AND CONCLUSION: No test item-related changes in parameters for fertility, early embryonic development, maternal function, and embryo-fetal development were observed during the study period. On the basis of these results, it was concluded that KGC-HJ3 did not have toxicological potential on developmental and reproductive functions. Therefore, no observed adverse effect levels of KGC-HJ3 for fertility, early embryonic development, maternal function, and embryo-fetal development is considered to be at least 2000 mg/kg/day.

A subchronic toxicity study of Radix Dipsaci water extract by oral administration in F344 rats.

Radix Dipsaci, the dried root of Dipsacus asperoides C.Y. Cheng & T.M.Ai, has therapeutic effects on various disorders, and in particular, bone and joint disease. Despite such ethnomedicinal benefits, there is very little information regarding its in vivo toxicity or adverse effects. This study was conducted to evaluate the potential toxicity of the Radix Dipsaci water Extract (RD-wE) by using F344 rats. The RD-wE was administered orally to rats at doses of 0, 125, 250, 500, 1000, and 2000 mg/kg body weight (bw)/day for 13 weeks. During the treatment period there were no mortalities attributed to RD-wE. Moreover, no toxic effects were observed with regard to body weight, clinical pathology (hematology, clinical biochemistry, and urinalysis), and anatomic pathology (gross findings, organ weight, and microscopic examination). The changes related to the treatment were excessive salivation at the mouth and soft feces, observed in male and female rats at 1000 or 2000 mg/kg bw/day, but these were not accompanied by any microscopic correlate or other pathophysiological changes. Based on these results, the oral no-observed-adverse-effect level of the RD-wE was considered to be 2000 mg/kg bw/day in both genders, although the target organs were not determined under the current experimental conditions.

Developmental toxicity assessment of the new turf herbicide, methiozolin ([5-(2,6-difluorobenzyl)oxymethyl-5-methyl-3,3(3-methylthiophen-2-yl)-1,2-isoxazoline]), in rabbits.

Methiozolin is a new herbicide to control annual bluegrass (Poa annua L.) and large crabgrass (Digitaria sanguinalis (L.) Scop.) in various turfgrasses. The potential of methiozolin to induce maternal and developmental toxicity was investigated in the pregnant New Zealand White Rabbits. Methiozolin was, at dose levels of 0, 125, 250 and 500 mg/kg/day, administered by oral gavage to artificially inseminated rabbits (25 females per group) from days 6 to 28 of gestation. All does were subjected to Cesarean section on day 29 of gestation. At 500 mg/kg/day, treatment-related toxicities including abortion (10/22), decreased mean body weight, weight gain, net body weight change, reduced food consumption and decreased fetal weight were observed. At 125 and 250 mg/kg/day, no signs of maternal and developmental toxicity were observed. There were no treatment-related external, visceral and skeletal abnormalities of fetuses at all doses tested. In the current experimental conditions, the no observed adverse effect levels (NOAELs) of methiozolin are considered to be 250 mg/kg/day for does and prenatal development.

Embryotoxicity and toxicokinetics of the antimalarial artesunate in rats.

This study was conducted to investigate the potential embryo-fetal toxicity and toxicokinetics of the antimalarial agent artesunate (ARTS) in Sprague-Dawley rats. Pregnant rats were administered ARTS daily from gestational day 6~15 via oral gavage, at test doses of 0, 2, 4, or 8 mg/kg (22 females per group). The fetuses were examined for external, visceral, and skeletal abnormalities on gestational day 20. With regard to the dams, there were no deaths, treatment-related clinical signs, changes in body weight, or food intake in any of the treatment groups. There were no treatment-related gross findings at necropsy in any treatment group. In the 8 mg/kg group, there was a decrease in gravid uterine weight and in the weight of female fetuses. There was also an increase in fetal deaths (primarily late resorptions) and an increase in post-implantation losses (37%) at 8 mg/kg. An increase in the incidence of visceral and skeletal variations at 4 and 8 mg/kg was observed. These defects included minor changes in the appearance of the kidney and thymus, as well as absent ribs or thoracic vertebrae. Toxicokinetics were assessed in a parallel study, using 4 mated females per group. Using liquid chromatography-mass spectrometry (LC-MS) analysis, the concentration of ARTS and its metabolite dihydroartemisinin (DHA) were quantified in plasma from rats on gestational days 5, 6, 10, and 15. Amniotic fluid was assayed for ARTS and DHA on gestational day 15. There was evidence of rapid conversion of ARTS to the metabolite DHA in maternal plasma, since ARTS could not be consistently detected in plasma at the three doses tested. ARTS and DHA were not detected in amniotic fluid at gestational day 15, indicating limited placental transfer of the two agents. The embryofetal no-observable-adverse-effect level (NOAEL) of the test item was considered to be 8 mg/kg/day for dams, and 2 mg/kg/day for embryo-fetal development.

Milk transfer and toxicokinetics of valproic Acid in lactating cynomolgus monkeys.

Studies on milk transfer of drugs in non-human primates (NHPs) are among the crucial components in the assessment of peri- and postnatal toxicity because of the similarity between NHPs and humans. To evaluate the milk transfer of valproic acid (VPA) in NHPs, the toxicokinetics of VPA, an antiepileptic drug, were studied in pregnant cynomolgus monkeys. VPA was administered once daily to pregnant cynomolgus monkeys at doses of 0, 30, 90, and 270 mg/kg by oral gavage from Day 100 of gestation (GD 100) to Day 31 of lactation (LD 31). Concentrations of VPA and its metabolite, 4-ene-VPA, in the maternal plasma on GD 100, GD 140, and LD 30, and concentrations of VPA and 4-ene-VPA in the offspring plasma and milk on LDs 30 and 31, respectively, were quantified using liquid chromatography tandem mass spectrometry (LC/MS/MS). After administration of a single oral dose of VPA to pregnant monkeys on GD 100, the concentrations of VPA and 4-ene-VPA were generally quantifiable in the plasma of all treatment groups up to 24 hr after administration, which showed that VPA was absorbed and that the monkeys were systemically exposed to VPA and 4-ene-VPA. After administration of multiple doses of VPA to the monkeys, VPA was detected in the pup's plasma and in milk taken on LD 30 and LD 31, respectively, which showed that VPA was transferred via milk, and the pup was exposed to VPA. Further, the concentration of VPA in the milk increased with an increase in the dose. Extremely low concentrations of 4-ene VPA were detected in the milk and in the pup plasma. In conclusion, pregnant monkeys were exposed to VPA and 4-ene-VPA after oral administration of VPA at doses of 30, 90, and 270 mg/kg/day from GD 100 to LD 31. VPA was transferred via milk, and the VPA exposure to the pup increased with an increase in the dose of VPA. The metabolite, 4-ene VPA, was present in extremely low concentrations (< 0.5 µg/ml) in the milk and in the pup plasma. In this study, we established methods to confirm milk transfer in NHPs, such as mating and diagnosis of pregnancy by examining gestational sac with ultrasonography, collection of milk and pup plasma and determination of toxicokinetics, using cynomolgus monkeys.

Toxicity assessment of Leonuri Herba aqueous extract orally administered to rats for 13 consecutive weeks.

ETHNOPHARMACOLOGICAL RELEVANCE: The Leonuri Herba has been traditionally used for women's disease in Asian countries. AIM OF THE STUDY: The objective of the present study was to evaluate the subchronic toxicity of Leonuri Herba aqueous extract in male and female F344 rats. MATERIAL AND METHODS: Leonuri Herba aqueous extract was administered orally once daily at dose levels of 0, 125, 250, 500, 1000 and 2000 mg/kg/day for 13 weeks. Toxicological assessment was performed including mortality, clinical signs, body and organ weights, food consumption, ophthalmology, urinalysis, hematology, serum chemistry, gross findings and histopathologic examination. RESULTS: There were no treatment related differences in clinical signs, urinalysis, hematology and serum chemistry, except for a histopathologic examination. The squamous cell hyperplasia in the forestomach was observed in both sexes of rats given 2000 mg/kg/day of Leonuri Herba aqueous extract. CONCLUSION: In conclusion, the NOAEL (No Observed Adverse Effect Level) for Leonuri Herba aqueous extract was determined as 1000 mg/kg/day in both sexes of rats under the present experimental conditions. And the acceptable daily intake value for Leonuri Herba aqueous extract was calculated to be 10mg/kg body weight per day using a safety factor of 100 to the NOAEL.

Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin.

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last)). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.

One-generation reproductive toxicity study of 2-methylbutane in Sprague-Dawley rats.

This study investigated the potential reproductive toxicity of 2-methylbutane in a one-generation reproductive toxicity study using Sprague-Dawley rats. A total of 24 male and female rats per group were given 2-methylbutane by gavage at 0, 100, 300, and 1000 mg/kg/day. Males were dosed for 10 weeks prior to mating and during mating. Females were dosed from 2 weeks before mating to day 21 of lactation. At 1000 mg/kg/day, both genders exhibited an increase in adrenal gland weight, however, a decrease in body weight gain and food intake, an increase in kidney weight, and an increased incidence of histopathological changes of the kidney were also observed in male rats. No treatment-related effects of 2-methylbutane were found in relation to the reproductive capacity of parental animals or the pre- and post-natal development of the F1 generation. There were no treatment-related effects in either gender at ≤ 300 mg/kg/day. Under these experimental conditions, the no-observed-adverse-effect level of 2-methylbutane was 300 mg/kg/day for general toxicity and 1000 mg/kg/day for reproductive capacity and pup development in rats.

Lack of a co-promotion effect of 60 Hz circularly polarized magnetic fields on spontaneous development of lymphoma in AKR mice.

The present study was conducted to investigate the possible effect of 60 Hz circularly polarized magnetic fields (MFs) as promoters of genetically initiated lymphoma in AKR mice. One hundred sixty female animals were divided into four different groups. They were exposed to four different intensities of circularly polarized MFs. Animals received exposure to 60 Hz circularly polarized MF at field strengths (rms-value) of 0 microT (sham control, T1, Group I), 5 microT(T2, Group II), 83.3 microT (T3, Group III), or 500 microT(T4, Group IV), for 21 h/day from the age of 4-6 weeks to the age of 44-46 weeks. There were no exposure-related changes in mean survival time, clinical signs, body weights, hematological values, micronucleus assay, gene expression arrays, analysis of apoptosis, and necropsy findings. At histopathological examination, lymphoma was seen in all the groups. The tumor incidence was 31/40(78%), 30/40(75%), 32/40(80%), and 31/40(78%) in sham control, 5, 83.3, and 500 microT groups, respectively. However, there were no differences in the tumor incidence between the sham control (T1) and circularly polarized MF exposure groups (T2-T4). In conclusion, there was no evidence that exposure to 60 Hz circularly polarized MF strengths up to 500 microT promoted lymphoma in AKR mice.

Placenta transfer and toxicokinetics of valproic Acid in pregnant cynomolgus monkeys.

Placenta transfer study in non-human primate (NHP) is one of the crucial components in the assessment of developmental toxicity because of the similarity between NHP and humans. To establish the method to determine placenta transfer in non-human primate, toxicokinetics of valproic acid (VPA) , a drug used to treat epilepsy in pregnant women, were determined in pregnant cynomolgus monkeys. After mating, pregnancy-proven females were daily administered with VPA at dose levels of 0, 20, 60 and 180 mg/kg by oral route during the organogenesis period from gestation day (GD) 20 to 50. Concentrations of VPA and its metabolite, 4-ene-VPA, in maternal plasma on GDs 20 and 50, and concentrations of VPA and 4-ene-VPA in placenta, amniotic fluid and fetus on GD 50 were analyzed using LC/MS/MS. Following single oral administration of VPA to pregnant monkeys, concentrations of VPA and 4-ene-VPA were generally quantifiable in the plasma from all treatment groups up to 4-24 hours post-dose, demonstrating that VPA was absorbed and the monkeys were systemically exposed to VPA and 4-ene-VPA. After repeated administration of VPA to the monkeys, VPA was detected in amniotic fluid, placenta and fetus from all treatment groups, demonstrating that VPA was transferred via placenta and the fetus was exposed to VPA, and the exposures were increased with increasing dose. Concentrations of 4-ene-VPA in amniotic fluid and fetus were below the limit of quantification, but small amount of 4-ene-VPA was detected in placenta. In conclusion,pregnant monkeys were exposed to VPA and 4-ene-VPA after oral administration of VPA at dose levels of 20, 60 and 180 mg/kg during the organogenesis period. VPA was transferred via placenta and the fetus was exposed to VPA with dose-dependent exposure. The metabolite, 4-ene VPA, was not detected in both amniotic fluid and fetus, but small amount of 4-ene-VPA was detected in placenta. These results demonstrated that proper procedures to investigate placenta transfer in NHP, such as mating and diagnosis of pregnancy via examining gestational sac with ultrasonography, collection of amniotic fluid, placenta and fetus after Caesarean section followed by adequate bioanalysis and toxicokinetic analysis, were established in this study using cynomolugus monkeys.

Pathological features of bone marrow transplantation-related toxicity in a mouse.

In this case report, we present a mock-transduced bone marrow (BM) transplantation in a mouse, which was found moribund and autopsied to evaluate pathogenesis. Macroscopically, red discoloration of systemic organs was observed. Hematological values revealed a decrease in white blood cells, red blood cells, hematocrit, hemoglobin, and platelets, but an increase in reticulocytes. In BM cytology, hematopoietic cell lines were severely depleted. Histopathologically, hemorrhage in the cerebellar parenchyma, hemosiderin deposition and hemorrhage in the heart, necrosis and telangiectasia in liver, pulmonary parenchymal cysts, spermatogenic germ cells necrosis, atrophy and hemorrhage in testis, oligospermia and hemorrhage in the epididymis, and atrophy of BM, thymus and spleen were observed. In conclusion, autoimmune-like complications such as hematological value change, BM dysplasia and systemic hemorrhage appear to be the lethal cause of the mouse transplanted with mock-transduced BM.

Combined repeated dose and reproductive/developmental toxicities of copper monochloride in rats.

This study investigated the combined repeated dose and reproductive/developmental toxicity of copper monochloride in rats. The test substance was administered once daily by gavage at 0, 1.3, 5, 20, or 80 mg/kg/day. Male rats were dosed for a total of 30 days beginning 14 days before mating. Female rats were dosed from 2 weeks before mating to day 3 of lactation throughout the mating and gestation period. At 80 mg/kg/day, deaths were observed in 3 out of 12 females. There was a dose-dependent increase in the incidence of clinical signs and a reduction in the food consumption. Hematological and serum biochemical investigations revealed a decrease in the red blood cell, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin (MCH), and serum total protein levels and an increase in the white blood cell and platelets in males, and a decrease in the MCH and an increase in the platelets in females. Histopathological examination showed an increased incidence of squamous cell hyperplasia of the stomach in both genders as well as increased hematopoiesis of the femur in males. There was an increase in the number of icteric and runt pups at birth. At 20 mg/kg/day, there was an increase in the incidence of clinical signs and squamous cell hyperplasia of the stomach in both genders. At 5 mg/kg/day, an increase in the incidence of squamous cell hyperplasia of the stomach was observed in females. There were no adverse effects in the lowest group in both genders. Based on these findings, the no-observed-adverse-effect levels of copper monochloride were concluded to be 5 mg/kg/day in male rats and 1.3 mg/kg/day in female rats for general toxicity and 20 mg/kg/day for reproductive/developmental toxicity.

Basic Principles of the Validation for Good Laboratory Practice Institutes.

Validation specifies and coordinates all relevant activities to ensure compliance with good laboratory practices (GLP) according to suitable international standards. This includes validation activities of past, present and future for the best possible actions to ensure the integrity of non-clinical laboratory data. Recently, validation has become increasingly important, not only in good manufacturing practice (GMP) institutions but also in GLP facilities. In accordance with the guideline for GLP regulations, all equipments used to generate, measure, or assess data should undergo validation to ensure that this equipment is of appropriate design and capacity and that it will consistently function as intended. Therefore, the implantation of validation processes is considered to be an essential step in a global institution. This review describes the procedures and documentations required for validation of GLP. It introduces basic elements such as the validation master plan, risk assessment, gap analysis, design qualification, installation qualification, operational qualification, performance qualification, calibration, traceability, and revalidation.

Lack of a co-promotion effect of 60 Hz rotating magnetic fields on N-ethyl-N-nitrosourea induced neurogenic tumors in F344 rats.

The present study was conducted to investigate the possible effect of 60 Hz magnetic fields as promoters of brain tumors initiated transplacentally by ethylnitrosourea (ENU) in F344 rats. One hundred twenty mated animals were divided into six different groups and exposed in utero on day 18 of gestation to a single intravenous dose of either Saline (vehicle control, Group I), or ENU 10 mg/kg (Groups II-VI). In the present study, a total of 480 offspring was used. The offspring in group II were given no further treatment while the offspring in Groups III-VI were exposed to four different intensities of magnetic fields. Animals received exposure to 60 Hz magnetic field at field strengths of 0 Tesla (sham control, T1, Group III), 5 muT (T2, Group IV), 83.3 muT (T3, Group V), or 500 muT (T4, Group VI), for 21 h/day from the age of 4 weeks to the age of 32 or 42 weeks. At histopathological examination, tumors of the nervous system were seen in all the ENU-treated groups. The tumor incidence of the ENU group at 32nd and 42nd week necropsy was higher than that of the vehicle control group. The incidence of glial tumors at 42nd week necropsy was higher than the 32nd week necropsy. However, there were no differences in the tumor incidence between the sham control (T1) and ENU + magnetic field exposure groups (T2-T4). In conclusion, there was no evidence that exposure of offspring to 60 Hz at magnetic field strengths up to 500 muT to the age of 32 or 42 weeks promoted ENU-initiated brain tumors in rats.

Lack of dominant lethality in mice following 1-bromopropane treatment.

1-Bromopropane (1-BP) is widely used in spray adhesives, precision cleaner, and degreaser. This study was conducted to investigate the potential of 1-BP to induce dominant lethality in mice. 1-BP was orally administered to males at doses of 300 and 600 mg/kg for 10 days before mating. Cyclophosphamide was used as a positive control (PC), which was administered intraperitoneally to males at 40 mg/kg for 5 days. The vehicle control (VC) group received corn oil only. Thereafter, males were mated with untreated females during six sequential mating periods of a week each. Males were sacrificed at the end of mating and so were the pregnant females on days 15-17 of gestation. Clinical signs, gross findings, mating index, gestation index, the numbers of corpora lutea, implantations, live fetuses, resorptions and dead fetuses, pre- and post-implantation losses, and dominant lethal mutation rate were examined. There were no treatment-related changes in clinical signs, gross findings, mating index, gestation index, number of corpora lutea and implantations, pre-implantation loss, live fetuses, resorptions, dead fetuses, post-implantation loss at any 1-BP doses tested. In the PC group, there were no treatment-related changes in mating index, gestation index, number of corpora lutea, and dead fetuses. However, a decrease in the number of implantations and an increase in pre-implantation loss were observed during the first 2 weeks as compared to those of the VC group. No treatment-related changes were observed in the third to sixth weeks. Increases in resorptions, fetal deaths and post-implantation loss, and a decrease in the number of live fetuses were observed in the first 3 weeks of the PC group compared to those of the VC group. However, no treatment-related changes were observed during the forth to sixth weeks. An increase in dominant lethal mutation rate was observed in 1-3 weeks of mating of the PC group, but there was no significant difference in 1-6 weeks of mating of the 1-BP treatment groups. In conclusion, 1-BP did not induce dominant lethality in mice. These results are in agreement with the report of Saito-Suzuki et al., demonstrating that no dominant lethality of 1-BP was observed in Sprague-Dawley rats.

Reproductive toxicity evaluation of a new camptothecin anticancer agent, CKD-602, in pregnant/lactating female rats and their offspring.

CKD-602 is a camptothecin anticancer agent that was recently developed by the Chong Kun Dang Pharmaceutical Co. (Seoul, Korea). This study examined the potential adverse effects of CKD-602 on pregnancy, delivery, and lactation in female Sprague-Dawley rats as well as on the pre- and postnatal development of their offspring. One hundred pregnant females were divided into four groups: three treatment groups and a control group. CKD-602 was administered once daily by intravenous bolus injection to female rats at doses of 0, 5.7, 17, or 51 microg/kg/day from gestational day 6, through to parturition and throughout the period of lactation up to weaning [lactational day (LD) 21]. All the dams were sacrificed on LD 22 after weaning. The clinical signs, mortality, body weight change, food consumption, physical development, and behavioral function were evaluated in their progeny. When the exposed offspring reached maturity (postnatal day 70), their reproductive performance was assessed. In the high-dose group, suppressed body weight and a decrease in the amount of food consumption were observed in the dams during both the gestation and lactation periods. An increase in the incidence of thymic atrophy, decreased liver and ovary weight, and an increase in the weight of the spleen were also observed in the dams at the scheduled necropsy. In addition, an increase in the number of stillborn and postnatal mortality, a decrease in the live litter size, and a delay in physical development were observed in the F1 offspring. Teratological examinations showed an increase in the incidence of congenital anomalies in both the F1 offspring and F2 fetuses. In the medium dose group, only slight maternal toxicity including suppressed body weight and decreased food consumption was observed. There were no treatment-related effects on the maternal function and pre- and postnatal development in the low dose group. The no-observed-adverse-effect level (NOAEL) of CKD-602 for the dams are considered to be 5.7 microg/kg/day, however, the NOAEL for their offspring are estimated to be 17 microg/kg/day.

Evaluation of the toxic potentials of a new camptothecin anticancer agent CKD-602 on fertility and early embryonic development in rats.

This study examined the potential adverse effects of a new camptothecin anticancer agent, CKD-602, on the fertility and early embryonic development of Sprague-Dawley rats. Ninety-six rats of each gender were divided into four groups: three treatment groups and a control group. CKD-602 was administered intravenously to male rats at 0, 4.7, 14, and 42 microg/kg from 63 days prior to mating until the end of the mating period, and to female rats from 14 days before mating until day 6 of gestation. All the males were sacrificed after the end of the 14-day mating period, while all the females were subjected to a caesarean section on day 15 of gestation. In the high dose group, a high incidence of hair loss was observed in both genders. A decrease in the level of food consumption, followed by a decrease in body weight gain was also observed in both genders. At the scheduled necropsy, the gross postmortem examinations revealed an increase in the incidence of thymic atrophy, paleness of the thoracic and abdominal organs in both genders and an increase in the serum testosterone concentration. In addition, there was a decrease in the thymus weight of the males and an increase in the liver, spleen, kidneys, lung, and heart weights of the females. There was an increase in the number of fetal deaths and post-implantation losses as well as a decrease in the litter size found at the caesarean section of the dams. No treatment-related effect on the histopathological findings, sexual cycle, pre-coital time, mating index, fertility index, pregnancy index, and sperm parameters was observed. There were no adverse effects on the general findings and reproductive performance of the parent animals and early embryonic development in the low and medium dose groups. Overall, the no-observed-adverse-effect levels (NOAELs) of CKD-602 are believed to be 14 microg/kg for both general toxicity and early embryonic development, and more than 42 microg/kg for the reproductive performance of the parent animals.

Embryotoxic effects of CKD-602, a new camptothecin anticancer agent, in rats.

CKD-602 is a newly developed camptothecin anticancer agent. Preclinical studies suggest that it may have greater antitumor activity and lower toxicity than other camptothecin anticancer agents. The potential of CKD-602 to induce embryotoxicity was investigated in the Sprague-Dawley rat. One hundred mated females (sperm in vaginal lavage=day 0) were distributed among three treatment groups and a control group. CKD-602 was administered intravenously at dose levels of 0, 5, 20 and 80 microg/kg/d to pregnant rats from days 6 to 15 of gestation. The vehicle control rats received an equivalent volume of 1 ml distilled water with d-mannitol 50mg and tartaric acid 0.06 mg. All dams were subjected to the caesarean section on day 20 of gestation. There were no signs of maternal toxicity or embryotoxicity at 5 microg/kg/d, but at 20 microg/kg/d, there was an increase in relative brain weight. At 80 microg/kg/d, reduced food intake, suppressed body weight and increased weight of spleen were observed in dams. An increase in the resorptions and dead fetuses, a decrease in litter size, fetal and placental weights were also found. In addition, various types of external, visceral and skeletal malformations occurred. Characteristic malformations included absent eye bulge, agnathia, dilated cerebral ventricle, anophthalmia, absent thoracic centrum, fused vertebral arch, fused rib, among others. Visceral and skeletal variations were observed. Retarded ossification of several skeletal districts and delayed ossification of sternebrae, metatarsals and sacrocaudal vertebrae were also observed. The results show that CKD-602 is embryotoxic and teratogenic at a minimally maternally toxic dose, i.e. at 80 microg/kg/d in rats. The no-observed-adverse-effect level (NOAEL) of CKD-602 for developmental toxicity was considered to be 20 microg/kg/d, however, the NOAEL for maternal toxicity was 5 microg/kg/d.

Effects of CKD-602, a new camptothecin anticancer agent, on pregnant does and embryo-fetal development in rabbits.

CKD-602 is a newly developed camptothecin anticancer agent. Preclinical studies suggest that it may have greater antitumor activity and lower toxicity than other camptothecin anticancer agents. The potential of CKD-602 to induce developmental toxicity was investigated in the New Zealand White rabbit. Seventy-two artificially inseminated females (artificial insemination=day 0) were distributed among three treatment groups and a control group. CKD-602 was at dose levels of 0, 0.024, 0.048, or 0.096 mg x kg(-1) x day(-1) administered intravenously to pregnant does from days 6 to 18 of gestation. All does were subjected to caesarean section on day 28 of gestation. At 0.096 mg x kg(-1) x day(-1), 2 cases of abortion and 3 cases of death in pregnant rabbits were found in late gestation. In addition, an increase in the embryonic resorptions and a decrease in the litter size were found. At 0.048 mg x kg(-1) x day(-1), a single doe aborted on gestational day 26. An increase in the embryonic resorptions and fetal morphological alterations and a decrease in the litter size were also found. There were no signs of maternal toxicity or developmental toxicity at 0.024 mg x kg(-1) x day(-1). The results show that 13-day repeated intravenous dose of CKD-602 during the major organogenetic period in rabbits produces increased incidence of abortion and death, increased number of embryonic resorptions and fetal morphological alterations, and decreased litter size at dose levels of above 0.048 mg x kg(-1) x day(-1). In the current experimental conditions, the no-observed-adverse-effect levels (NOAELs) of CKD-602 are considered to be 0.048 mg x kg(-1) x day(-1) for does and 0.024 mg x kg(-1) x day(-1) for embryo-fetal development.

Identification of potential biomarkers of genotoxicity and carcinogenicity in L5178Y mouse lymphoma cells by cDNA microarray analysis.

In the present study, cDNA microarray analyses were performed with mouse cDNA chips in order to evaluate similarities and differences in the gene expression profiles for compounds differing in their genotoxic and carcinogenic potential. Eight test substances were evaluated, two each from four classes of compounds: genotoxic carcinogens (1,2-dibromoethane and glycidol), genotoxic noncarcinogens (8-hydroxyquinoline and emodin), nongenotoxic carcinogens (methyl carbamate and o-nitrotoluene), and nongenotoxic noncarcinogens (D-mannitol and 1,2-dichlorobenzene). Quadruplicate hybridization experiments were performed in order to identify a set of genes with significant expression changes for these four classes of substances. Twelve genes were consistently altered more than twofold by the genotoxic noncarcinogens while four genes were consistently regulated by the nongenotoxic carcinogens. One gene (Trp63) was identified whose expression was upregulated by all four genotoxic substances regardless of the presence or absence of carcinogenicity; this finding, however, was not confirmed by quantitative real-time RT-PCR. RT-PCR did confirm the change in expression of 9 of 15 genes (60%) identified by microarray analysis. Interestingly, the downregulated genes were least likely to be validated by real-time RT-PCR. Those genes showing more than a twofold change in expression level in response to at least one substance were further analyzed with hierarchical clustering after category assignment of each gene according to its main cellular function. Clustering revealed differences in the gene expression profiles between the genotoxic and nongenotoxic substances for genes involved in cell cycle control, the stress response, and the immune response. However, no clustering specific to all four carcinogenic substances was observed in any of the functional categories. Taken together, these results suggest that gene expression profiling in mouse lymphoma cells can provide valuable information for the evaluation of potential genotoxicity but may have limitations in predicting carcinogenicity.